EPID 525 Lecture 2
Manual and automated methods
for organism identification
Microscopy
• Magnification
– enhancement of size using ocular and objective lenses.
• Ocular: eyepiece (10X)
• Objective: 4X – 100X
– allows for visualization of bacteria, fungi, and parasites, not viruses
• Resolution
– ability to distinguish two objects as distinct
– resolving power is closest distance between two objects
– immersion oil is added when using 100X objective to prevent light
scatter
• Contrast
– use stains to enhance visualization; allow organism to stand out
from background
Staining techniques
• make slide by smear, drop, or cytocentrifuge
• dry, then fix by heat (flame, 10 min at 60ºC)
or fix by methanol (95% 1min)
• Gram stain
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Crystal violet: primary stain
Gram’s iodine: mordant/fixative
Acetone-ethanol: decolorizer
Safranin: counterstain
http://www.sp.uconn.edu/~terry/229sp02/lectures/Lect2.html
Neisseria gonorrhea - Gram stain
http://www.cdc.gov/STD/LabGuidelines/default.htm
Wound specimen - Gram stain
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Oral specimen - Gram stain
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Sputum specimen Gram stain—Streptococcus pneumoniae
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Sputum specimen Gram stain—mixed oral flora
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Sputum—cystic fibrosis patient, encapsulated gram negative rods
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Staining techniques
• Acid-fast stains
– for staining of organisms with high degree of fatty (mycolic)
acids—waxy
– render the cells resistant to decolorization: “acid-fast”
– Mycobacterium sp., Nocardia sp., Cryptosporidium sp. are
acid-fast
– Procedure
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Ziehl-Neelsen: heat drives in primary stain (carbolfuchsin)
Kinyoun: higher conc. of phenol does not require heat
Decolorize with acid-alcohol
Counterstain with methylene blue or malachite green
A Gram stain (left) of the abscess shows thin, gram positive rods in chains.
An acid fast stain (right) was also positive.
http://pathhsw5m54.ucsf.edu/overview/bacteria3.html
Sputum specimen—Acid fast stain
http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
Acid fast stain demonstrating chording
Microscopy
• Phase Contrast Microscopy
– shift in light allows visualization of organism; can visualize viable
organisms
• Fluorescent Microscopy
– certain dyes (fluorochromes) give off light when excited
(fluorescence)
– color of light depends on the dye and the filters used
– Staining techniques
• Fluorochroming: direct chemical interaction with organism
– Acridine orange: stains nucleic acid; useful for cell-wall deficient organisms
– Auramine-rhodamine: bind to mycolic acids in nearly all Mycobacteria
– calcofluor white: binds to chitin in cell walls of fungi
• Immunofluorescence: fluorochrome is bound to an antibody; can
detect/identify specific organisms
http://aeneary.myweb.uga.edu/lepto_files/image013.gif
WBC
nuclei
Bacteria
Staphylococci, acridine orange stain
http://www.med.sc.edu:85/fox/strep-staph.htm
Mycobacterium – auramine stain
http://www.lung.ca/tb/abouttb/what/causes_tb.html
Yeast—calcofluor white
http://www.med.sc.edu:85/mycology/mycology-3.htm
Mould—calcofluor white
Influenza virus infected cells, fluorescent antibody stain
Culture and isolation of bacteria
•
Principles of Cultivation
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Nutritional requirements
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General concepts
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non-fastidious: simple requirements for growth
fastidious: complex, unusual, or unique
requirements for growth
Phases of growth media
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solid  agar; boil to dissolve, solidifies at 50ºC
liquid, broth
• Media classifications and functions
– Enrichment
• used to enhance growth of specific organisms
– Supportive
• support growth of most non-fastidious organisms
– Selective
• contains agents that inhibit the growth of all agents
except that being sought (dyes, bile salts, alcohols,
acids, antibiotics)
– Differential
• contains factor(s) that allow certain organisms to exhibit
different metabolic characteristics
• Types of artificial media
– Brain-heart infusion
• nutritionally rich supportive media used in broths, blood culture
systems and susceptibility testing
– Sheep blood agar
• supportive media containing 5% sheep blood for visualization of
hemolysis
– Chocolate agar
• same as sheep blood agar except blood has been
“chocolatized” RBCs lysed by heating; releases X (hemin) and
V (NAD) factors for Neisseria and Haemophilus
– MacConkey agar
• selective for Gram-negative rods (GNRs) because of crystal
violet and bile salts; differential due to lactose, fermenters lower
pH changing neutral red indicator pink/red
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Types of artificial media
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Hektoen enteric agar
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Columbia colistin-nalidixic acid (CNA) agar
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Columbia agar base, sheep blood, colistin and nalidixic acid;
selective isolation of gram-positive cocci
Thayer-Martin agar
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contains bile salts and dyes (bromothymol blue and acid
fuchsin) to inhibit non-pathogenic GNRs; non pathogens
ferment lactose changing BTB to orange; pathogens
Salmonella and Shigella are clear; ferric ammonium citrate
detects H2S production of Salmonella (black colonies)
CAP with antibiotics (colistin inhibits gram neg, vancomycin
inhibits gram pos, nystatin inhibits yeast); for N. gonorrhoeae
and N. meningitidis; Martin-Lewis has similar function but
different antibiotics
Preparation of artificial media
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Sterilization
•
autoclave: pressurized steam at 121ºC for 15-30 min.
http://science.nhmccd.edu/biol/wellmeyer/media/media.htm
Chocolate agar
Uninoculated
Haemophilus
http://www.hardydiagnostics.com/catalog2/hugo/ChocolateAgar.htm
MacConkey agar
http://science.nhmccd.edu/biol/wellmeyer/media/media.htm
Hektoen agar
http://medic.med.uth.tmc.edu/path/hekto.htm
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Environmental requirements
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Oxygen and Carbon dioxide availability
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aerobic: room air
facultative: aerobic or anaerobic
microaerophilic: reduced oxygen tension
anaerobic
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strict or aerotolerant
capnophilic: increased C02 (5-10%)
Temperature
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35-37ºC
30ºC
cold
42ºC
• Bacterial Cultivation
– Isolation of bacteria from specimens
• streaking for isolation
• streaking for quantitation
– Evaluation of colony morphologies
• Type of media supporting growth
• Relative quantities of each colony type
• Colony characteristics
– colony form: pinpoint, circular, filamentous, irregular
– colony elevation: flat, raised, convex
– colony margin: smooth, irregular
• Gram stain and subcultures
– sterile loop, isolated colonies
Conventional methods for ID
• Principles of Identification
– ID using genotypic criteria
– ID using phenotypic criteria
• Microscopic morphology and staining
characteristics
• Colony morphology
• Environmental requirements for growth
• Resistance or susceptibility to antimicrobial
agents
– bacitracin, novobicin, vancomycin
• Nutritional requirements and metabolic capabilities
– Single enzyme tests
• Catalase: H2O2 + catalase = O2 and H20; differentiates
Staphylococcus v. Streptococcus, Listeria and corynebacteria
v. other non spore forming gram-positive bacilli
• Oxidase: detection of cytochrome oxidase that participates in
nitrate metabolism; Pseudomonas, Aeromonas, Neisseria
• Indole: tryptophanase degrades tryptophan into pyruvic acid,
ammonia, and indole; indole is detected by aldehyde indicator;
presumptive id for E. coli
• Urease: hydrolyzes urea into ammonia, water and CO2;
increase pH changes causes bright pink color of indicator
• PYR: hydrolysis of PYR, indicator turns pink; Group A Strep
and enterococci are +
– Tests for presence of metabolic pathways
• Oxidation and fermentation: oxidation of glucose requires
oxygen, fermentation does not; pH decreases causing yellow
color
• Amino acid degredation: detection of amino acid decarboxylase
enzymes
O-F glucose media
http://academic.mwsc.edu/jcbaker/bio390sec01/bio390_laboratory_study_images.htm
• Principles of Phenotype-based ID schemes
– Selection and inoculation of ID test battery
• Type of bacteria to be identified
• Clinical significance of isolate
• Availability of reliable testing methods
– Incubation for substrate utilization
• Conventional ID
• Rapid ID
– Detection of metabolic activity
• Colorimetry: pH change of indicators
• Fluorescence: release of fluorophore from substrate or changes
in fluorescence due to pH changes
• Turbiditiy: growth or no-growth
– Analysis of metabolic profiles
• ID databases
• Use of databases to ID unknowns
– Confidence in ID
API strips – bioMerieux
• Commercial ID systems
– Advantages and examples of commercial
systems
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ARIS (Trek)
MicroScan (Dade-Behring/Seimens)
Phoenix (Becton-Dickinson)
Vitek (bioMérieux)
VITEK 2
bioMérieux
Microscan
Dade Behring
Immunochemical methods for ID
• Principles of immunochemical methods
– Particle agglutination
• Latex agglutination
– Immunofluorescent assays
• Direct immunofluorescence assay (DFA)
• Indirect immunofluorescence assay (IFA)
• Enzyme immunoassays
– Solid-phase immunoassays
• Membrane bound immunoassays
• Immunochromatographic assays
– Optical immunoassays
Latex agglutination
Lateral flow immunoassay
Serologic methods for diagnosis
• Features of the Immune Response
– Characteristics of antibodies
• Features of immune response useful in diagnostic testing
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Acute v. anamnestic response
IgM v. IgG
IgM can’t cross placenta
immunocompetent v. immunocompromised
• Interpretation of serologic tests
– single v. paired sera; rare pathogen
– 4-fold rise in titer v. qualitative testing
– cross reactivity (herpes viruses, heterophile Abs,
pregnancy)
• Principles of Serologic Test Methods
– Methods for antibody detection
• Direct whole pathogen agglutination assays
– pos patient sera causes organism to clump
• Particle agglutination tests
– latex beads or RBCs coated with Ag
• Flocculation tests
– RPR – precipitation of soluble Ag with Ab
» charcoal particles coated with cardiolipin-lecithin binds
reagin
• ELISAs
• IFAs
– organism/antigen on slides; patient Ab detected with
fluorescent secondary Ab
• Western blots
ELISA
Western Blot